title: Global analysis of ion dependence unveils hidden steps in DNA binding and bending by integration host factor.
abstract: Proteins that recognize and bind to specific sites on DNA often distort the DNA at these sites. The rates at which these DNA distortions occur are considered to be important in the ability of these proteins to discriminate between specific and nonspecific sites. These rates have proven difficult to measure for most protein-DNA complexes in part because of the difficulty in separating the kinetics of unimolecular conformational rearrangements (DNA bending and kinking) from the kinetics of bimolecular complex association and dissociation. A notable exception is the Integration Host Factor (IHF), a eubacterial architectural protein involved in chromosomal compaction and DNA recombination, which binds with subnanomolar affinity to specific DNA sites and bends them into sharp U-turns. The unimolecular DNA bending kinetics has been resolved using both stopped-flow and laser temperature-jump perturbation. Here we expand our investigation by presenting a global analysis of the ionic strength dependence of specific binding affinity and relaxation kinetics of an IHF-DNA complex. This analysis enables us to obtain each of the underlying elementary rates (DNA bending/unbending and protein-DNA association/dissociation), and their ionic strength dependence, even under conditions where the two processes are coupled. Our analysis indicates interesting differences in the ionic strength dependence of the bi- versus unimolecular steps. At moderate [KCl] (100-500 mM), nearly all the ionic strength dependence to the overall equilibrium binding affinity appears in the bimolecular association/dissociation of an initial, presumably weakly bent, encounter complex, with a slope SK(bi) ≈ 8 describing the loglog-dependence of the equilibrium constant to form this complex on [KCl]. In contrast, the unimolecular equilibrium constant to form the fully wrapped specific complex from the initial complex is nearly independent of [KCl], with SK(uni) < 0.5. This result is counterintuitive because there are at least twice as many ionic protein-DNA contacts in the fully wrapped complex than in the weakly bent intermediate. The following picture emerges from this analysis: in the bimolecular step, the observed [KCl]-dependence is consistent with the number of DNA counterions expected to be released when IHF binds nonspecifically to DNA whereas in the unimolecular reorganization step, the weak [KCl]-dependence suggests that two effects cancel one another. On one hand, formation of additional protein-DNA contacts in the fully wrapped complex releases bound counterions into bulk solution, which is entropically favored by decreasing [salt]. On the other hand, formation of the fully wrapped complex also releases tightly bound water molecules, which is osmotically favored by increasing [salt]. More generally, our global analysis strategy is applicable to other protein-DNA complexes, and opens up the possibility of measuring DNA bending rates in complexes where the unimolecular and bimolecular steps are not easily separable.
authors.affiliation: Department of Physics, University of Illinois at Chicago, Chicago, Illinois 60607, USA.
authors.forename: Paula
authors.initials: P
authors.lastname: Vivas
authors.forename: Yogambigai
authors.initials: Y
authors.lastname: Velmurugu
authors.forename: Serguei V
authors.initials: SV
authors.lastname: Kuznetsov
authors.forename: Phoebe A
authors.initials: PA
authors.lastname: Rice
authors.forename: Anjum
authors.initials: A
authors.lastname: Ansari
mesh_terms: D001665:Binding Sites; D004247:DNA / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer; D034982:Integration Host Factors / Q000737:chemistry*; D007700:Kinetics; D007834:Lasers; D008958:Models, Molecular; D009010:Monte Carlo Method; D009994:Osmolar Concentration; D011189:Potassium Chloride / Q000737:chemistry; D013696:Temperature; D013816:Thermodynamics
chemical_list: D034982:Integration Host Factors; D011189:Potassium Chloride; D004247:DNA